The promyelocyte-like leukemia cell line HL-60 can be induced to differentiate terminally in vitro. Treatment with agents such as dimethyl sulfoxide (Me.sub.2 SO) and retinoic acid (RA) results in the expression of a granulocytic phenotype (Collins, et al., 1977; Breitman, et al., 1980), while exposure to 1,25-dihydroxyvitamin D.sub.3 (1,25-(OH).sub.2 D.sub.3) or phorbol 12-myristate 13-acetate (PMA) leads to the expression of a monocytic-macrophagic phenotype (Bar-Shavit, et al., 1983; Huberman and Callagham, 1979; McCarthy, et al., 1983; Rovera, et al., 1979). A common effect of all these inducers is that HL-60 cells undergo growth arrest while they terminally differentiate. A number of genes are known to be regulated during HL-60 differentiation: c-fms is induced when cells differentiate along the monocyte-macrophage pathway (Sariban, et al., 1985); c-fos is rapidly induced by PMA but not by 1,25-(OH).sub.2 D.sub.3, Me.sub.2 SO, and RA (Muller, et al., 1985); c-myc is down-regulated by PMA and 1,25-(OH).sub.2 D.sub.3 (Einat, et al., 1985; Reitsma, et al., 1983) as well as Me.sub.2 SO and RA (Einat, et al., 1985; Westin, et al., 1982).
We have recently derived and analyzed a clonal variant of HL-60, 1F10, that permits us to study several intermediate steps in HL-60 differentiation (Cayre, et al., 1987). 1F10 cells treated with PMA or 1,25-(OH).sub.2 D.sub.3 are arrested at discrete steps in differentiation (Cayre, et al., 1987).
These cells are still blastic and resemble a promyelocyte. They are morphologically similar to the original HL-60 cells, grow exponentially, and do not exhibit phenotypic characteristics of differentiating cells. Complete maturation and growth arrest of the 1F10 cells are attained when 1,25-(OH).sub.2 D.sub.3 and PMA are used simultaneously. When the two inducers are added sequentially, cells must be treated first with 1,25-(OH).sub.2 D.sub.3 to fully differentiate (Cayre, et al., 1987). This and the study of genes such as c-myc (Reitsma, et al., 1983), pD3-137 (Solomon, et al., 1988), and c-fms (Sariban, et al., 1985), which are similarly regulated by 1,25-(OH).sub.2 D.sub.3 in both HL-60 and 1F10 cells, suggested that the 1,25-(OH).sub.2 D.sub.3 -treated 1F10 cells are arrested at an immature step in differentiation (Cayre, et al., 1987; Solomon, et al., 1988).
To identify genes that are regulated at discrete steps in HL-60 differentiation, cDNA subtraction was applied to the 1F10 system (Solomon, et al., 1988). By this method, we have isolated a cDNA clone encoding a novel serine protease, myeloblastin. Myeloblastin mRNA is down-regulated by granulocytic and monocytic inducers of HL-60 differentiation. In the absence of inducer, myeloblastin mRNA is regulated by serum. We have used an antisense oligodeoxynucleotide to inhibit myeloblastin expression. This inhibition results in proliferation arrest and differentiation of the leukemic cells.
As shown by deduced amino acid sequence analysis and DFP (diisopropylfluorophosphate) labelling, myeloblastin is a serine protease. Because protease inhibitors affect the growth of normal and transformed cells, earlier inventigators have proposed a role for proteases in the regulation of cell growth (Gibson, et al., 1984; Sullivan and Quigley, 1986). A series of cell surface-related proteolytic events involving serine proteases with thrombin-like activity are required for cell proliferation and differentiation (for review, see Carrell, 1988). These events are controlled by surface-associated inhibitors of serine proteases (Baker, et al., 1986). For example, one of the protease nexins has been identified as the natural factor that promotes the outgrowth of neurites from neuronal cells (Gloor, et al., 1986). More recently, other investigators have cloned a cDNA from HL-60 cells that has homology to members of a family of Kunitz-type serine protease inhibitors and that is also a potential promoter of neurite growth (Tanzi, et al., 1988; Carrell, 1988). These Kunitz-type inhibitors have specificity for several serine proteases including elastase (for review, see Carrell, 1988).
Myeloblastin shares amino acid sequence homology with HuNE (Human neutrophil elastase). Similar to myeloblastin mRNA, HuNE mRNA is expressed in both HL-60 and U937 cells (Takahashi, et al., 1988). As is the case for myeloblastin mRNA, the HuNE mRNA is down-regulated by PMA in HL-60 cells (Takahashi, et al., 1988). A major difference between HuNE and myeloblastin is that myeloblastin mRNA is down-regulated while HuNE mRNA is up-regulated when HL-60 cells are exposed to Me.sub.2 SO (Takahashi, et al., 1988). The functional relevance, if any, of amino acid homologies between HuNE and myeloblastin is unclear at present.
Regulation of myeloblastin has similarities with regulation of c-myc. A remarkable feature of myeloblastin is that it is down-regulated by both monocytic and granulocytic inducers of HL-60 differentiation. This down-regulation by several inducers is consistent with the fact that this serine protease is associated with proliferation arrest--which always accompanies HL-60 differentiation (Collins, et al., 1977; Huberman and Callagham, 1979; Rovera, et al., 1979; Breitman, et al., 1980; Bar-Shavit, et al., 1983; McCarthy, et al., 1983)--and is reminiscent of the down-regulation of c-myc mRNA during differentiation of HL-60 cells. As is the case for myeloblastin mRNA, induced differentiation along either the granulocytic or the monocytic pathway results in a profound decrease in c-myc mRNA levels (Sariban, et al., 1985; Reitsma, et al., 1983; Westin, et al., 1982); c-myc is transcriptionally controlled by 1,25-(OH).sub.2 D.sub.3 (Simpson, et al., 1987) and is involved in cell proliferation (Kelly, et al., 1983; Kaczmarek, et al., 1985). As is the case for myeloblastin mRNA, c-myc mRNA is down-regulated at different times with different inducers (Reitsma, et al., 1983; Sariban, et al., 1985; Siebenlist, et al., 1988). The fact that myeloblastin mRNA is down-regulated at different times using different inducers is consistent with previous reports that showed that Me.sub.2 SO, PMA, retinoic acid, and 1,25(OH).sub.2 D.sub.3 at concentrations similar to those used in our study did not differentiate HL-60 cells at the same rate (Collins, et al., 1978; Rovera, et al., 1979; Breitman, et al., 1980; McCarthy, et al., 1983). The inhibition of c-myc expression by specific antisense oligodeoxynmucleotides oligodeoxynucleotides results in inhibition of proliferation and induction of differentiation of HL-60 cells (Heikkila, et al., 1987; Wickstrom, et al., 1988; Holt, et al., 1988).
Other genes that are regulated during HL-60 differentiation, such as c-myc and c-fos, can be induced in response to serum. Because myeloblastin mRNA is induced in response to serum stimulation of HL-60 cells, we propose that myeloblastin is a member of a restricted family of genes that are associated with proliferation and are regulated during HL-60 differentiation. Since reduction of c-myc expression may not be obligatory or sufficient for growth arrest to occur (Shen-Ong, et al., 1987; Cayre, et al., 1987), it will be of interest to know whether myeloblastin and c-myc are interdependent for controlling proliferation and differentiation of HL-60 cells. It will also be of interest to explore whether similar serum and transcription factors regulate both myeloblastin and c-myc expression.
Myeloblastin is a novel protein not disclosed in any earlier references. Detecting its expression in undifferentiated leukemia cells provides an improved means of determining whether the cells are lymphoblastic or monocytic. Also, levels of myeloblastin expression provide information about the onset and development of leukemia that is useful in prognosis.
In addition, the use of antisense oligonucleotides complementary to myeloblastin mRNA provide a therapeutic treatment for leukemia by down-regulating myeloblastin expression in leukemia cells, which results in a reversal of their abnormal behavior. This method represents a distinct improvement over chemotherapy in that it causes no damage to normal cells. Natural myeloblastin inhibitors for therapeutic use also have advantages over chemotherapy in specificity and safety. Wickstrom, et al, 1988 and 1989, disclose antisense oligonucleotides complementary to the oncogene c-myc, and Agrawal, et al., 1989, disclose antisense oligodeoxynucleotides complementary to HIV RNA. However, none of these references disclose antisense oligonucleotides complementary to myeloblastin mRNA.